The eventual application of pluripotent based cell replacement therapies will require the development of methods that enable large scale culture and differentiation conditions that are compliant with regulatory guidelines.
In the United States, the Food and Drug Administration (FDA) has issued guidance in the form of Draft Guidance for Reviewers: Instructions and Template for Chemistry, Manufacturing, and Control (CMC) Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications (INDs); and in the 21 CFR §1270 and §1271 regulations. In Europe, the requirement for cell therapy products is outlined in several directives and guidelines that are pertinent to human embryonic stem (hES) cells. See Directive 2004/23/EC, Commission Directives 2006/17/EC and 2006/86/E, EU Regulation 1394/2007, guideline EMEA/CHMP/410869, which are herein incorporated by reference in their entireties. Each country has certain regulatory standards for quality and safety including donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Regulations establish guidelines for development, manufacturing and quality control as well as non-clinical and clinical development of cell-based medicinal products. See also C. Unger et al. (2008), Human Molecular Genetics 17(R1):R48-R53.
Regulatory guidelines present many hurdles for manufacturing-scale production of stem cells and products thereof. For example, to maintain pluripotent cells in an undifferentiated state in vitro, research studies still employ animal products such as mouse embryonic fibroblast (MEF) feeders and fetal bovine serum (FBS). Still other cell culture conditions for maintaining pluripotentcy contain serum replacers, such as KnockOut™ Serum Replacer (KSR; Invitrogen), which although more defined, still contains a complex crude mixture containing unknown compounds as well as bovine serum albumin (BSA) or lipid-rich albumin fraction of bovine serum (AlbuMAX). Further, serum batches vary in their activity and therefore their capability of maintaining pluripotent undifferentiated cell cultures. Hence, the replacement of bovine serum with non-animal derived (“xeno-free”) defined components is preferred for GMP production of pluripotent cells.
Applicants previously determined that activation of the IGF1, Insulin, ERBB2 and ERBB3 receptors was a hallmark of proliferating hES cells. IGF1R/IR activation could be attributed to IGF1 in serum or microgram/ml concentrations of insulin, for example, in serum replacer or N2/B27 cell culture supplements in a range of growth conditions. These activities provide a strong PI3 kinase/AKT signal, and equivalent activation could be achieved with lower concentrations of LR3-IGF1, an IGF1 analog. See McLean et al., 2007 Stem Cells 25, 29-38. Phosphorylation of ERBB2/3 was consistent with the activity of an EGF family member, heregulin/neuregulin, in MEF-CM. See Wang et al., 2007 Blood 110, 4111-9 Inhibition of IGF1R and ERBB2 impacted self-renewal of hES cell, as did small molecule inhibitors of Activin and FGF signaling. See Robins and Schulz, 2009, “Novel methods of Stem Cell Culture and Maintenance: Media and extra cellular matrix requirements for large scale ESC growth.” In Emerging Technology Platforms for Stem Cells (eds. U. Lakshmipathy J. D. Chesnut and B. Thyagarajan, pp. 251-247) Hoboken. John Wiley & Sons Inc.; and Vallier et al., 2005 J Cell Sci 118, 4495-509). This defined medium was developed and termed DC-HAIF, which consisted of DMEM/F12, non essential amino acids, trace elements, ascorbic acid, β-mercaptoethanol, Penicillin/Streptomycin (optional), with the only proteins being caprylic acid extracted fatty acid-free BSA, transferrin, and recombinant Heregulin-1β (H), Activin A (A), LR3-IGF1 (I), and FGF2 (F). DC-HAIF supported long term maintenance of pluripotent hES cells, as well as single cell passaging and scaled expansion of hES cell using ACCUTASE™. See Robins and Schulz (2009) supra; Wang et al., supra. A batch-tested commercial formulation of DC-HAIF is available from under license to Life Technologies and sold under the trade name StemPro® hES cell SFM.
Still there remains a need to identify alternative and/or additional signaling pathways with critical functions in pluripotent stem cells, e.g. hES and iPS cells, and that are able to be used for therapeutic purposes, wherein the culture compositions are defined and/or produced to GMP standard.